Pharmaceutically purified intact bacterial minicells

ABSTRACT

The present invention provides a method for purifying bacterial minicells that involves subjecting a sample containing minicells to density gradient centrifugation in a biologically compatible medium. The method optionally includes a preliminary differential centrifugation step and one or more filtration steps. The invention also provides a method for purifying bacterial minicells in which a sample containing minicells is subjected to a condition that induces parent bacterial cells to adopt a filamentous form, followed by filtration of the sample to separate minicells from parent bacterial cells. The inventive methods optionally include one or more steps to remove endotoxin from purified minicell preparations, and/or treatment of purified minicell preparations with an antibiotic. Additionally, the invention provides purified minicell preparations, prepared according to the foregoing methods, and containing fewer than about 1 contaminating parent bacterial cell per 10 7 , 10 8 , 10 9 , 10 10 , or 10 11  minicells.

CLAIM TO CONVENTION PRIORITY

This application is a continuation of U.S. application Ser. No.11/691,698, filed Mar. 27, 2007, now U.S. Pat. No. 8,003,091 which is adivisional of U.S. application Ser. No. 10/602,021, filed Jun. 24, 2003now U.S. Pat. No. 7,611,885. The subject matter of each application isincorporated herein by reference in the entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a pharmaceutically compatible methodfor purifying intact bacterial minicells.

A minicell is an anucleate form of an E. coli or other bacterial cell,engendered by a disturbance in the coordination, during binary fission,of cell division with DNA segregation. Prokaryotic chromosomalreplication is linked to normal binary fission, which involves mid-cell,septum formation. In E. coli, for example, mutation of min genes, suchas minCD, can remove the inhibition of septum formation at the cellpoles during cell division, resulting in production of a normal daughtercell and an anulceate minicell (de Boer et al., 1992; Raskin & de Boer,1999; Hu & Lutkenhaus, 1999; Harry, 2001).

In addition to min operon mutations, anucleate minicells also aregenerated following a range of other genetic rearrangements or mutationsthat affect septum formation, for example in the divIVB1 in B. subtilis(Reeve and Cornett, 1975; Levin et al., 1992). Minicells also can beformed following a perturbation in the levels of gene expression ofproteins involved in cell division/chromosome segregation. For example,overexpression of minE leads to polar division and production ofminicells. Similarly, chromosome-less minicells may result from defectsin chromosome segregation, for example the smc mutation in Bacillussubtilis (Britton et al., 1998), spoOJ deletion in B. subtilis (Iretonet al., 1994), mukB mutation in E. coli (Hiraga et al., 1989), ana parCmutation in E. coli (Stewart and D'Ari, 1992). Gene products may besupplied in trans. When over-expressed from a high-copy number plasmid,for example, CafA may enhance the rate of cell division and/or inhibitchromosome partitioning after replication (Okada et al., 1994);resulting in formation of chained cells and anucleate minicells (Wachiet al., 1989; Okada et al., 1993).

Minicells are distinct from other small vesicles that are generated andreleased spontaneously in certain situations and, in contrast tominicells, are not due to specific genetic rearrangements or episomalgene expression. Exemplary of such other vesicles are bacterial blebs,which are small membrane vesicles (Dorward et al., 1989). Blebs havebeen observed in several bacterial species from Agrobacterium, Bacillus,Bordetella, Escherichia, Neisseria, Pseudomonas, Salmonella andShigella, for example. Bacterial blebs can be produced, for instance,through manipulation of the growth environment (Katsui et al., 1982) andthrough the use of exogenous membrane-destabilizing agents (Matsuzaki etal., 1997).

Because plasmid replication within prokaryotic cells is independent ofchromosomal replication, plasmids can segregate into both normaldaughter cells and minicells during the aberrant cell division describedabove. Thus, minicells derived from recombinant min E. coli carrysignificant numbers of plasmid copies, with all of the bacterialcellular components except for chromosomes, and have been used as suchin studying plasmid-encoded gene expression in vitro. Sec Brahmbhatt(1987), Harlow et al. (1995), and Kihara et al. (1996). Brahmbhatt(1987) demonstrated, for example, that E. coli minicells can carryrecombinant plasmids with DNA inserts as large as 20 kb, absent anychromosomal DNA, and can express nine or more recombinant proteinssimultaneously.

A recent patent application, PCT/IB02/04632 (incorporated entirelyherein by reference), described recombinant, intact minicells containingtherapeutic nucleic acid molecules. Such minicells are effective vectorsfor delivering oligonucleotides and polynucleotides to host cells invitro and in vivo. Accordingly, they are particularly useful forintroducing nucleic acid molecules that, upon transcription and/ortranslation, function to ameliorate or otherwise treat a disease ormodify a trait associated with a particular cell type, tissue or organof a subject.

In vivo minicell applications generally require minicell preparations ofa high purity, particularly with respect to live parent bacteria, freeendotoxin and Cellular debris (including membrane fragments, nucleicacids and intracellular components) that might elicit an inflammatoryresponse in an immunized host. Moreover, the use of minicells incommercial pharmaceutical products will require methods for purifyingminicells to approved international pharmaceutical standards. To thisend, conventional methods of minicell purification generally areunsatisfactory.

Conventional techniques entail (a) low speed centrifugation, to reducethe bio-burden of parent cells, and (h) differential rate sedimentationin a gradient of glycerol, sucrose or percoll. An initial differential,low speed centrifugation typically reduces parental cells by as much as100-fold, while leaving 50% to 70% of minicells in the supernatantfluid. Two subsequent cycles of differential rate sedimentation thenyield minicell preparations having a purity of about 1 vegetative cellper 10⁶-10⁷ minicells. Such conventional methods are reviewed by Frazer& Curtiss (1975), and are described by Reeve (1979), Clark-Curtiss &Curtiss (1983), and U.S. Pat. No. 4,311,797 (to Khachatourians).

The purity achieved by conventional purification methods may not beadequate for all in vivo applications, some of which may require dosesgreater than 10⁶ minicells, or even 10¹⁰ minicells. At theaforementioned contamination ratio, this would translate into 10,000live parent cells per dose. Such a contamination level could be fatal,particularly in immuno-compromised patients such; as cancer and AIDSpatients. For example, the ID₅₀ (infectious dose in 50% Of infectedpeople) for Shigella dysenteriae, Salmonella enteritidis and Listeriamonocytogenes organisms is approximately 10, 1,000 and 10 respectively.Moreover, previous studies have reported that the level of parental cellcontamination varies with different bacterial strains (Clarke-Curtissand Curtiss, 1983). In that regard, gene therapy applications describedin PCT/IB02/04632 may employ minicells derived from a range of mutantGram-negative and Gram-positive bacterial strains, and would requireminicells that are essentially free of live parent bacterial cellcontamination. Thus, conventional minicell purification methods do notpermit quality control for cGMP (current good manufacturing practice)manufacture of biopharmaceutical doses of minicells.

As an additional drawback, the gradient formation media (percoll,sucrose and glycerol) employed by conventional purification methods areincompatible with in vivo uses. Percoll is toxic and, hence, isrestricted to “research purposes only” contexts. Sucrose imparts a highosmolarity to gradients that can cause physiological changes inminicells. Indeed, the present inventors have determined that minicellsundergo an osmotic shock in sucrose gradients and, as a consequence,become structurally deformed. Glycerol is highly viscous and difficultto remove completely from the minicell suspensions. Accordingly,although these density gradient media effectively separate cells andcellular organelles or components, they are not suitable for separatingbiological cells that are destined for clinical use in humans.

Several approaches have been developed to improve conventional minicellpurification techniques. One approach employs parent cells that carry achromosomal recA mutation, and treatment with low doses of Ultra Violet(UV) radiation (Sancar et al., 1979). The rationale of this approach isthat UV radiation will preferentially degrade chromosomal DNA because ofits large target size, as opposed to smaller plasmid DNA. However,recombinant minicells used for gene therapy and vaccine applicationsmust be free of any mutation, and non-specific mutagenesis methods suchas UV radiation would not ensure that all plasmid DNAs remainun-mutated.

Another approach to improve minicell purification operates byinhibiting, bacterial cell wall synthesis, such as by using ampicillinor cycloserine, or by starving diaminopimelic acid (DAP)-requiringstrains of DAP (Clarke-Curtiss and Curtiss, 1983). This approach alsosuffers from several drawbacks, however. First, many recombinantplasmids used for gene therapy will carry an ampicillin resistancemarker, which renders parent cells carrying the plasmid ampicillinresistant. Second, many in-vivo minicell applications will employminicells derived from a range of different bacterial species, many ofwhich may not be susceptible to DAP-requiring mutations. Third, anylarge-scale use of antibiotics is undesirable due to the attendant risksof generating antibiotic-resistant bacteria.

Recently, a novel approach for purifying minicells that addresses theabove-mentioned concerns was reported (PCT/IB02/04632). The novel methodcombines cross-flow filtration (feed flow is parallel to a membranesurface; Forbes, 1987) and dead-end filtration (feed flow isperpendicular to the membrane surface) to achieve a minicell purity thatexceeds 10⁻⁷ (i.e., fewer than one parent cell per 10⁷ minicells), andeven 10⁻⁹. Optionally, the filtration combination can be preceded by adifferential centrifugation, at low centrifugal force, to remove someportion of the bacterial cells and thereby enrich the supernatant forminicells.

Although this filtration procedure overcomes the drawbacks associatedwith conventional minicell purification techniques, it also haslimitations. Foremost, cross-flow filtration results in considerableloss of minicells, which adds cost to the manufacturing process.Additionally, minicell preparations obtained by the filtration procedurecontain some bacterial endotoxin, which causes a mild shock whenadministered in viva. Finally, minicell purity varies from batch tobatch when the filtration methods are employed.

Therefore, a need remains for methods of purifying bacterial minicellsthat maximize minicell yield and purity, while employing biologicallycompatible media.

To address these and other needs, the present invention provides amethod for purifying bacterial minicells that involves subjecting asample containing minicells to density gradient centrifugation in abiologically compatible medium. The method optionally includes apreliminary differential centrifugation step.

The present invention also provides a method for purifying bacterialminicells that combines density gradient centrifugation in abiologically compatible medium with filtration.

In another aspect, the present invention provides a minicellpurification method in which a sample containing minicells is subjectedto a condition that induces parent bacterial cells to adopt afilamentous form, followed by filtration of the sample to separateminicells from parent bacterial cells.

In yet another aspect, the present invention provides a minicellpurification method that includes (a) subjecting a sample containingminicells to density gradient centrifugation in a biologicallycompatible medium, (b) subjecting the sample to a condition that inducesparent bacterial cells to adopt a filamentous form, then (c) filteringthe sample to obtain a purified minicell preparation.

The inventive methods optionally include one or more steps to removeendotoxin from purified minicell preparations, and/or treatment ofpurified minicell preparations with an antibiotic.

Finally, the present invention provides purified minicell preparations,prepared according to the foregoing methods, and Containing fewer thanabout 1 contaminating parent bacterial cell per 10⁷, 10⁸, 10⁹, 10¹⁰ or10¹¹ minicells

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one way in which minicell purification techniques ofpresent invention can be integrated with other minicell purificationprocedures.

FIG. 2 shows Scanning Electron Micrographs of S. typhimuriumminCDE-strain bacteria (range of different sizes) and minicells derivedfrom the strain. (A) shows a small sized parent bacterium (1.1 um long)and a minicell (0.4 um diameter); (B) shows a larger parent bacterium(1.32 um long), (C) shows an even larger parent bacterium (1.6 um long),and (D) shows a mixture of parent bacteria and minicells, where theformer range in length from 1 um to 4 um.

FIG. 3A shows the filamentation of parent S. typhimurium minCDE-strainbacteria following incubation with various NaCl concentrations forvarious times.

FIG. 3B shows fluorescence microscope images comparing the sizes of S.typhimurium minCDE-strain bacteria incubated in growth media for 4 hrsin the absence of NaCl (left side image) and of bacterial filamentsformed after 4 hrs incubation in the presence of 5% NaCl (right sideimage).

FIG. 4A shows the filamentation of parent E. coli minCDE-strain bacteriafollowing incubation with various NaCl concentrations for various times.

FIG. 4B shows fluorescence microscope images comparing the sizes of E.coli minCDE-strain bacteria incubated in growth media for 4 hrs in theabsence of NaCl (left side image) and of bacterial filaments formedafter 4 hrs incubation in the presence of 5% NaCl (right side image).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present inventors have determined that the use of biologicallycompatible media improves conventional minicell purification. In thisregard, they have observed that commonly used density gradient media,while effective at separating minicells from contaminants, often haveadverse effects on minicells. For example, conventional methods commonlyemploy 30% sucrose gradients and require two to three repeated sucrosegradient purifications to achieve adequate purity. This exposesminicells to high osmotic pressure for up to two hours, likely causingosmotic shock to the minicells. The present inventors have found thatsucrose-gradient purified minicells often are significantly deformedrelative to minicells purified by other means. Presumably, the deformityresults from membrane destabilization, which allows excess fluid intothe minicells. Such membrane destabilization, and its attendant increasein membrane porosity, also amid allow cytosol contents, includingtherapeutic nucleic acids to leak out of the minicells.

In one aspect, therefore, the present invention contemplates a minicellpurification method that comprises separating minicells from parentbacterial cells and other contaminants via density gradientcentrifugation in a biologically compatible medium. Aftercentrifugation, a minicell band is collected from the gradient, and,optionally, the minicells may be subjected to further rounds of densitygradient centrifugation to maximize purity. The method may furtherinclude a preliminary step of performing differential centrifugation onthe minicell-containing sample. When performed at low centrifugal force,differential centrifugation will remove some portion of parent bacterialcells, thereby enriching the supernatant for minicells.

“Biologically compatible media,” used in this context, refers to mediathat do not adversely affect minicell physiology or morphology.Preferably, biologically compatible media also do not adversely affecthost cell physiology, or host organism physiology. The meaning of“biologically compatible” is therefore contextual. For example, aparticular medium may be biologically compatible to one type ofminicell, but toxic to another. Preferably biologically compatible mediaare both isotonic and non-toxic.

OptiPrep™ (Axis-Shield PLC, Dundee, Scotland), which is a sterile 60%(w/v) solution of iodixanol (5,5′-[(2-hydroxy-1-3propanediyl)-bis(acetylamino)] bis[N,N′-bis(2,3-dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide]) inwater, constitutes one highly preferred example of a biologicallycompatible medium. Researchers have extensively utilized OptiPrep™ andother similar density gradient media for purifying mammalian cells andorganelles, as well as membrane vesicles, viruses, proteins, nucleicacids and lipoproteins. These uses are reviewed in Density GradientMedia. Applications and Products 2002, Axis-Shield PLC, Dundee,Scotland. Such media were not previously employed, however, to purifybacterially-derived minicells. Indeed, prior to the present inventors'observation that other media adversely affect minicell physiology andmorphology, a need for biologically compatible media to purify minicellswas not even recognized.

With OptiPrep™ it is possible to use either preformed gradients, or toform a gradient in situ by centrifugation (self-generating gradient).Preformed gradients can be continuous or discontinuous gradients.Preformed gradients of OptiPrep™ can be formed by layering solutions ofthe desired concentrations into a centrifuge tube and allowing thesolutions to diffuse by sealing the top of the tube and laying it on itsside during diffusion. The preparation of isoosmotic density gradientswith OptiPrep™ depends upon preparing gradient solutions by dilution ofOptiPrep™solution with an appropriate diluent solution. Selection of adiluent solution and osmotic balancers is well within the ordinary skillor practitioners.

In another aspect, the present invention combines density gradientcentrifugation in a biologically compatible medium with filtering steps.For example, density gradient centrifugation can be incorporated into aserial filtration process, as exemplified in FIG. 1. One such serialfiltration process is described in PCT/IB02/04632. Briefly, that processcombines cross-flow filtration (feed flow is parallel to a membranesurface) and dead-end filtration (feed flow is perpendicular to amembrane surface). Optionally, this combination can be preceded by adifferential centrifugation, at low centrifugal force, to remove someportion of parent bacterial cells and thereby enrich the supernatant forminicells. Also optionally, the combination can be followed by anantibiotic treatment to kill residual parent bacterial cells.

Cross-flow filtration, depending on the filter pore size, can separateminicells from larger contaminants such as parent bacterial cells, andfrom smaller contaminants such as bacterial blebs, free endotoxin,nucleic acids, cellular debris and excess liquid. To separate minicellsfrom larger contaminants, the nominal pore Size of cross-flow filtersshould allow minicells to permeate through the filters, but not largebacterial cells. A 0.45 μm pore size is preferred for this purposebecause minicells are approximately 0.4 μm in diameter, whilst bacterialcells are larger. To separate minicells from smaller contaminants, thenominal pore size of cross-flow filters should allow smallercontaminants to permeate through the filters, but not minicells. A 0.2μm pore size is preferred for this purpose because bacterial blebs rangein diameter from 0.05 μm to 0.2 μm, and the other smaller contaminantsare less than 0.2 μm.

Effective application of cross-flow filtration in this context typicallyentails at least one step involving a larger pore size, around 0.45 μm,followed by at least one step with a smaller pore size, around 0.2 μm.Between or during serial cross-flow filtration steps, diafiltration maybe performed to maximize recovery of minicells. In the diafiltration,volume is held constant and ultrafiltration membranes are used to retaindesired particles (minicells, in this case), while undesirable smallersolutes and particles are removed.

The use of cross-flow filtration accommodates suspensions carrying heavyloads of particulate matter, such as bacterial cultures, which may carryloads of 10¹¹ to 10¹³ bacterial and minicell populations per liter ofculture. To minimize filter fouling and the consequent loss ofminicells, the bacterial/minicell culture may be diluted, preferably5-fold to 10-fold. Dilutions also permit use of appropriately low pumppressure and flow rate.

To remove residual parent bacterial cells remaining after cross-flowfiltration, dead-end filtration may be performed. For this purpose, theuse of at least one dead-end filtration, employing a pore size of about0.45 μm, is preferred.

In one embodiment, a minicell purification method combines densitygradient centrifugation through a biologically compatible medium with afiltration step that employs at least one filter with a pore size lessthan or equal to about 0.2 μm.

In another embodiment, a minicell purification method combines densitygradient centrifugation through a biologically compatible medium with adead-end filtration step employing a filter with a pore size of about0.45 μm.

The present inventors also have discovered that inducing parentbacterial cells to adopt a filamentous form, prior to filtration,significantly improves minicell purification. Because minicells andparent bacterial cells have the same diameter (average of 0.4 um) somebacterial cells can permeate a filter pore that barely accommodates aminicell (e.g., 0.45 μm cross-flow or dead-end filter pores), eventhough the length of bacterial cells is at least 1 μm. This occurs whenan oblong bacterial cell lodges itself perpendicular to a filter.However, bacterial cell filaments, consisting of bacterial cells joinedend-to-end cannot penetrate such filters.

Thus, another aspect of the invention entails inducing contaminatingparent bacterial cells to form filaments prior to filtration. This isaccomplished by subjecting a minicell suspension to environmentalconditions that induce a stress response in parent cells. Suchconditions are well known to those skilled in the art, and includeanaerobic conditions, nutrient limiting conditions and abnormal osmoticconditions. Hypertonic media are particularly useful for inducingfilamentation. In one example, a minicell suspension can be supplementedwith Trypticase Soy Broth (growth medium) that contains 5% sodiumchloride (stress inducer). Under such stress-inducing conditions, cellsfail to fully separate during cell division, and form long bacterialfilaments consisting of multiple cells.

Preferred embodiments of the invention exploit bacterial filamentationto increase minicell purity. Thus, in one aspect, the invention providesa minicell purification method that includes the steps of (a) subjectinga sample containing minicells to density gradient centrifugation in abiologically compatible medium, and (b) subjecting the sample containingminicells to a condition that induces parent bacterial cells to adopt afilamentous form, followed by (c) filtering the sample to obtain apurified minicell preparation.

The present inventors have further discovered that the removal ofendotoxin improves minicell preparations. In in vivo mouse studies, theyobserved a mild shock resulting from the use of minicell preparationscontaining residual endotoxins. Thus, useful minicell preparationspreferably are substantially free from endotoxins.

Methods for removing endotoxins are well-known in the art. One exemplarymethod utilizes magnetic beads (for example, Dynabeads™; Dynal biotech,Oslo, Norway) coated with anti-Lipid A antibodies. Antibody coatedmagnetic beads can be mixed with a minicell suspension in a tube, andincubated to allow antibody to bind to free lipopolysaccharide (LPS) viaits Lipid A portion. The tube carrying the suspension is then placed ina magnetic stand to immobilize the anti-Lipid A-LPS complexed magneticbeads, and the minicells are collected. Multiple cycles of incubationwith fresh beads can be performed to achieve the desired level ofpurity. Monoclonal antibodies that bind to epitopes found in thedeep-core polysaccharide part of LPS also are useful for removing freeendotoxin. The deep-core polysaccharide part of LPS is not thought to beexposed on bacterial membrane surfaces. Therefore, antibodies directedagainst this part of LPS should not bind to bacterial cell-bound LPS.Prior to use, such antibodies should be tested to ensure that they donot cross-react with cell-surface exposed components of LPS.

Due to the potential for bacterial endotoxins to cause adverse sideeffects, preferred minicell purification methods include one or moresteps to remove them. Thus, in one aspect, the invention provides aminicell purification method that employs a density gradientcentrifugation step in a biologically compatible medium, followed by oneor more steps to remove endotoxin from the resulting enriched minicellpreparation. More preferably, the method further includes one or morefiltration steps, as described above.

The minicell purification techniques described herein may be employed invarious combinations to obtain a preparation of a desired purity.Preferred methods include a combination of density gradientcentrifugation and filtration. Preferred methods also includestress-induced filamentation of parent bacterial cells followed byfiltration, and removal of endotoxin from minicell preparations. Oneexample of a method (schematically depicted in FIG. 1) that employs allof these techniques is as follows:

Step A: Differential centrifugation of a minicell producing bacterialcell culture. This step, which may be performed at 2000 g for about 20minutes, removes most parent bacterial Cells, while leaving minicells inthe supernatant.

Step B: Density gradient centrifugation using an isotonic and non-toxicdensity gradient medium. This step separates minicells from manycontaminants, including parent bacterial cells, with minimal loss ofminicells. Preferably, this step is repeated within a purificationmethod.

Step C: Cross-flow filtration through a 0.45 μm filter to further reduceparent bacterial cell contamination.

Step D: Stress-induced filamentation of residual parent bacterial cells.This may be accomplished by subjecting the minicell suspension to any ofseveral, stress-inducing environmental conditions.

Step E: Antibiotic treatment to kill parent bacterial cells.

Step F: Cross-flow filtration to remove small contaminants, such asmembrane blebs, membrane fragments, bacterial debris, nucleic acids,media components and so forth, and to concentrate the minicells. A 0.2μm filter may be employed to separate minicells from small contaminants,and a 0.1 μm filter may be employed to concentrate minicells.

Step G: Dead-end filtration to eliminate filamentous dead bacterialcells. A 0.45 um filter may be employed for this step.

Step H: Removal of endotoxin from the minicell preparation. Anti-Lipid Acoated magnetic beads may be employed for this Step.

Those skilled in the art can implement variations of these steps andincorporate additional purification steps, consistent with theprinciples outlined herein.

The foregoing methods for purifying, bacterial minicells providepurified minicell preparations useful for in vivo applications such asthose described in PCT/IB02/04632. These preparations contain fewer thanabout 1 contaminating parent bacterial cell per 10⁷ minicells,preferably fewer than about 1 contaminating parent bacterial cell per10⁸ minicells, more preferably fewer than about 1 contaminating parentbacterial cell per 10⁹ minicells, even more preferably fewer than about1 contaminating parent bacterial cell per 10¹⁰ minicells, and yet morepreferably fewer than about 1 contaminating parent bacterial cell per10¹¹ minicells. Most preferably, any contaminating parent bacterialcells are dead, and these preparations do not contain any live parentbacterial cells. Moreover, these preparations are substantially free ofendotoxins.

Reference to the following, illustrative examples will help to provide amore complete understanding of the invention.

EXAMPLE 1 Inconsistency of Filtration without the Inventive Techniques

This example illustrates that the use of filtration to purify minicells,without the inventive techniques, can produce inconsistent results.

Minicell-producing mutant bacterial strains of S. typhimurium, E. coliand Shigella flexneri are analyzed by Scanning Electron Microscopy (SEM)to determine the size of the bacterial-cells and minicells. For HighResolution Scanning Electron Microscopy the following method isfollowed. Bacterial cultures are grown in Trypticase Soy Broth (TSB)(BBL brand purchased from Bacto Labs, Liverpool, NSW, Australia). Thebroth is prepared according to the manufacturer's instructions at 30gm/l, and autoclaved at 121° C. for 15 minutes. Liquid culture is grownovernight in a shaking incubator at 37° C. To change solutions the cellsare centrifuged at 13,000 rpm for 20 minutes, the supernatant isdiscarded, and the cells are resuspended in the new reagent (describedbelow) using a vortex mixer. This washes ions and biomaterials off thecells and leaves them suspended in a small volume of distilled water.The sequence of reagents is (a) 1 ml of distilled water-repellet, (b) 1ml of distilled water-resuspend, (c) deposit 250 μl on a clean brassspecimen plate, (d) dry overnight at 30° C., (e) coat just beforemicroscopy with 2 nm of chromium metal deposited in a Xenosput cleanvacuum sputter coater. The coated specimens are examined using anHitachi S-900 Field Emission Scanning Electron microscope using a beamenergy of 3 kilovolts (University of New South Wales, NSW, Australia).Digital images at different magnifications are recorded using anImageSlave digitizer.

The results show (representative images of S. typhimurium minCDE-strainare shown in FIGS. 2A-D) that parent bacterial cells range in lengthfrom 0.9 um to 4 μm and 0.4 μm to 0.5 μm in width. Following filtrationsteps outlined on the left side of FIG. 1, some batches show residualbacterial Contamination. The contaminating bacteria are small in size,i.e., about 0.9 um in length. This indicates that some small sizedbacteria that are approximately the same width as minicells (FIG. 2A)leak through the 0.45 μm cross-flow and dead-end filters.

EXAMPLE 2 Elimination of Small Bacteria: Conversion into BacterialFilaments

This Example demonstrates that inducing bacteria to filament prior tofiltration improves minicell purification processes.

A study is designed to address the problem described in Example 1, bymaking the residual small-sized parent bacteria substantially largerthan the 0.45 μm pore size of a dead-end filter. Stress-inducingconditions in a bacterial growth environment can prevent completeseparation during bacterial cell division, resulting in bacterialfilaments.

The study demonstrates that hypertonic bacterial growth media(stress-inducer) reliably induce filamentation of minicell-producingbacterial strains of S. typhimurium and E. coli. All bacteria are grownfrom glycerol stocks maintained at −80° C. S. typhimurium and E. colistrains are grown in Trypticase Soy Broth (TSB) (BBL brand purchasedfrom Bacto Labs, Liverpool, NSW, Australia). It is prepared according tothe manufacturer's instructions at 30 gm/l, and autoclaved at 121° C.for 15 minutes. Liquid culture is grown in a shaking incubator at 37° C.Overnight bacterial culture is diluted 1:5,000 in fresh TSB and grownuntil OD_(600nm) reaches 0.2. The culture is divided into ten 5 mlaliquots in sterile vials, and pre-autoclaved sterile NaCl is added toeach vial to yield final NaCl concentrations (w/v) of 0% (control), 2%,3%, 4.5%, 5%, 5.5%, 6%, 7% and 8%. The cultures are incubated staticallyat 37° C. and samples are obtained at 2 hrs, 4 hrs, 8 hrs and 24 hrs. Azero hour control sample is also obtained for microscopy. The samplesare centrifuged at 13,200 rpm and the bacterial/minicell pellets areresuspended in distilled water. A drop of each sample is placed on aglass slide, air dried and heat fixed. Each sample is Gram-stained usinga 95% alcohol wash followed by Gram Safranin flood for 1 min. and awater wash. The slides are visualized using the Leica Model DMLB lightmicroscope with image analysis by means of a Leica DC camera and LeicaIM image management software. Samples are viewed at 40× or oil immersionat 100× magnification.

The above-experiments are repeated four times to determine reliabilityof results and variations with a series of controls also are performed.

The results show (FIGS. 3A-B and 4A-B) that with increasing NaClconcentration, the bacterial cells form filaments comprising two totwenty coccobacilli stuck end-to-end. Within the range of 2% to 3% NaClconcentrations, filamentation is variable (FIGS. 3A and 4A), becauseseveral bacterial cells do not form filaments even after longerincubation periods. However, at 4% to 5% NaCl, the bacterial cellsreliably turn into filaments (FIGS. 3B and 4B). The optimum incubationperiod for filamentation at 4% to 5% NaCl is about 4 hrs, and furtherincubation up to 24 hrs is not generally necessary. Higher saltconcentrations of 5.5% to 8% decrease filament formation. Preliminarystudies to determine a viable bacterial count of each sample by dilutionplating on TSB agar plates suggests that significant numbers ofbacterial cells are killed at higher salt concentrations (5.5% to 8%NaCl), a potential reason why decreased filamentation is observed atthese NaCl concentrations.

A definitive study of the effect of the various NaCl concentrations onthe bacterial cell viability is performed out using the LIVE/DEADBacLight Bacterial Viability Kit (Molecular Probes, Eugene, Oreg., USA).The kit employs two nucleic acid stains, the green-fluorescent SYTO® 9stain and the red-fluorescent propidium iodide stain. These stainsdiffer in their ability to penetrate healthy bacterial cells. SYTO 9stain labels both live and dead bacteria. In contrast, propidium iodide(PI) penetrates only bacteria with damaged membranes, reducing SYTO 9fluorescence when both dyes are present. Thus, live bacteria with intactmembranes fluoresce green, while dead bacteria with damaged membranesfluoresce red. The above-described experiment on salt-inducedfilamentation is repeated, and 0 hr, 2 hr, 4 hr, 8 hr and 24 hr samplesfor the various NaCl concentrations are obtained. The samples arecentrifuged at 13,200 rpm, supernatant discarded and bacterial/minicellpellet is resuspended in 100 μl of BSG. 0.5 ml of a 50/50 mix of SYTO9/PI is added to each sample and incubated for 1.5 min. The samples arecentrifuged at 13,200 rpm, supernatant discarded and pellets areresuspended in 100 μl of distilled water. A drop of each sample isplaced on a glass slide, air dried and covered with a drop of BacLightMounting Oil. Each sample is visualized using the Leica Model DMLB lightmicroscope with image analysis by means of a Leica DC camera and Leicadigital image acquisition software. Samples are viewed at 40× or oilimmersion at 100× magnification.

The results show (color photos not shown) that at NaCl concentrations of5.5% and higher, significant numbers of bacterial cells fluoresce red(dead cells) and at NaCl concentrations of 7% and 8%, almost all of thebacterial Cells are dead within 2 hrs of incubation. This result showsthat 4% to 5% NaCl for an incubation time of 4 hrs is the maximum limitto achieve filamentation. After 2 hrs of incubation, the live bacterialcells turn into filaments. However as the incubation time increases thefilaments fluoresce red, suggesting that even 4% to 5% NaCl issufficient stress for the bacterial cells and that they begin to dieafter a few generations of growth. Since this stress appears to inhibitcomplete separation during bacterial cell division, it is sufficient topermit the formation of bacterial filaments. This data also explains whyfilamentation is not achieved at higher salt concentration: the stressis toxic, inhibiting bacterial growth and cell division, and causingcell death.

CITED PUBLICATIONS

-   Brahmbhatt, “Cloning and molecular characterization of the rfb gene    cluster of Salmonella typhimurium;” Ph.D. Thesis, University of    Adelaide, Australia (1987).-   Britton et al., “Characterization of a prokaryotic SMC protein    involved in chromosome partitioning,” Genes Dev. 12: 1254 (1998).-   Clark-Curtiss & Curtiss, “Analysis of recombinant DNA using    Escherichia coli minicells,” Methods Enzymol. 101: 347 (1983).-   de Boer et al., “Roles of MinC and MinD in the site-specific    separation block mediated by the MinCDE system of Escherichia    coli,” J. Bacteriol. 174: 63 (1992).-   Dorward et al., “Export and intercellular transfer of DNA via    membrane blebs of Neisseria gonorrhoeae,” J. Bacteriol. 171: 2499    (1989).-   Forbes, “Crossflow microfiltration,” Australian J. Biotechnology 1:    30 (1987).-   Frazer & Curtiss, “Production, properties and utility of bacterial    minicells,” Curr Top Microbiol. Immunol. 69: 1 (1975).-   Harlow et al., “Cloning and characterization of the gsk gene    encoding guanosine kinase of Escherichia coli,” J. Bacteriol. 177:    2236 (1995).-   Harry, “Bacterial cell division: Regulating Z-ring formation,” Mol.    Microbiol. 40: 795 (2001).-   Hiraga et al., “Chromosome partitioning in Escherichia coli: novel    mutants producing anucleate cells,” J. Bacteriol. 171: 1496 (1989).-   Hu & Lutkenhaus, “Topological regulation of cell division in    Escherichia coli involves rapid pole to pole oscillation of the    division inhibitor MinC under the control of MinD and MinE,” Mol.    Microbiol. 34: 82 (1999).-   Ireton et al., “spo0J is required for normal chromosome segregation    as well as the initiation of sporulation in Bacillus subtilis,” J.    Bacteriol. 176: 5320 (1994).-   Katsui et al., “Heat-induced blebbing and vesiculation of the outer    membrane of Escherichia coli,” J. Bacteriol. 151: 1523 (1982).-   Kihara et al., “Analysis of a FliM-FliN flagellar switch fusion    mutant of Salmonella typhimurium,” J. Bacteriol. 178: 4582 (1996).-   Levin at al., “Identification of Bacillus subtilis genes for septum    placement and shape determination,” J. Bacteriol. 174: 6717 (1992).-   Matsuzaki et al., “Interactions of an antimicrobial peptide,    magainin 2, with outer and inner membranes of Gram-negative    bacteria,” Biochim Biophys. Acta. 1327: 119 (1997).-   Okada et al., “Possible function of the cytoplasmic axial filaments    in chromosomal segregation and cellular division of Escherichia    coli,” Sci. Prog. 77: 253 (1993-94).-   Okada et al., “Cytoplasmic axial filaments in Escherichia coli    cells: possible function in the mechanism of chromosome segregation    and cell division,” J. Bacteriol. 176: 917 (1994).-   PCT/IB02/04632, Intact minicells as vectors for DNA transfer and    gene therapy in vitro and in vivo.-   Raskin & de Boer, “MinDE-dependent pole-to-pole oscillation of    division inhibitor MinC in Escherichia coli,” J. Bacteriol. 181:    6419 (1999).-   Reeve, “Use of minicells for bacteriophage-directed polypeptide    synthesis,” Methods Enzymol. 68: 493 (1979).-   Reeve & Cornett, “Bacteriophage SPO1-induced macromolecular    synthesis in minicells of Bacillus subtilis,” J. Virol. 15: 1308    (1975).-   Sancar, et al., “Simple method for identification of plasmid-coded    proteins,” J. Bacteriol. 137: 692 (1979).-   Stewart & D'Ari, “Genetic and morphological characterization of an    Escherichia coli chromosome segregation mutant,” J. Bacteriol. 174:    4513 (1992).-   Wachi et al., “New mre genes mreC and mreD, responsible for    formation of the rod shape of Escherichia coli cells,” J. Bacteriol.    171: 6511 (1989).

The invention claimed is:
 1. A pharmaceutical composition consistingessentially of at least 10⁹ intact bacterial minicells, wherein saidbacterial minicells are derived from Gram-positive or Gram-negativebacteria that generate free endotoxin and are approximately 0.4 μm indiameter, and wherein said composition (i) contains fewer than about 1contaminating parent bacterial cell per 10⁹ minicells and (ii) issubstantially free of membrane blebs, cellular debris, and freeendotoxin.
 2. The composition of claim 1, wherein said compositioncontains fewer than about 1 contaminating parent bacterial cell per 10¹⁰minicells.
 3. The composition of claim 1, wherein said compositioncontains fewer than about 1 contaminating parent bacterial cell per 10¹¹minicells.
 4. The composition of claim 1, wherein said compositioncomprises at least 10¹⁰ minicells.
 5. The composition of claim 1,wherein said bacterial minicells are derived from B. subtilis.
 6. Thecomposition of claim 1, wherein said free endotoxin is freelipopolysaccharide.